Az ozmotikus stresszválasz szabályozása magasabbrendű növényekben. = Regulation of osmotic stress responses in higher plants

Pályázatunkban egy új genetikai rendszert dolgoztunk ki, amely alkalmas a stressz jelátvitelben szerepet játszó növényi gének azonosítására. RT-PCR módszerrel jellemeztük több stressz indukált Arabidopsis gén aktivitását különböző stressz és homonkezelés után. A gének 5? promoter régióját megklónoztuk, és promoter nélküli luciferáz (LUC) illetve zöld fluorescens protein (GFP) riporter génekhez kapcsoltuk. A riporter gének aktivitását transzgenikus Arabidopsis növényekben tanulmányoztuk. Az új szabályozó faktorok azonosítása érdekében egy Arabidopsis cDNS könyvtárat hoztunk létre a pER8-GW expressziós vektorban, ami ösztradiol által indukálható expressziós kazettát hordoz. A cDNS könyvtár segítségével egy transzgenikus Arabidopsis növény populációt hoztunk létre. A transzgenikus növényeket só rezisztenciára, ABA érzékenységre illetve a már korábban beépített riporter gén aktivitásának megváltozására teszteltük. Több olyan Arabidopsis vonalat sikerült azonosítani, amelyekben az ösztradiol adása megnövekedett só vagy ABA toleranciával, illetve a riporter gén aktivitásával járt együtt. A C38-33 vonalban megemelkedett só toleranciát kaptunk a beépült cDNS transzkripciójának aktiválásával. A cDNS egy új, S1 domén-t tartalmazó fehérjét kódol. Az ADH-121 vonalban egy AP típusú transzkripció faktort azonosítottunk, ami képes volt az ADH-LUC riporter gén kontrukció aktiválására a külső környezeti tényezőktől függetlenül. | We have developed a genetic system to identify new regulatory factors, controlling stress responses in higher plants, namely in Arabidopsis. Using quantitative RT-PCR, we have characterized the expression of several stress-responsive genes in different conditions and hormonal treatments. The 5? promoter sequences of 5 stress-induced genes have been cloned and fused to promoterless reporter genes, such as the firefly luciferase (LUC) or the green fluorescence protein (GFP). Activity of the reporter gene constructs was characterized in transgenic Arabidopsis plants, using non-destructive assays. In order to identify new regulatory factors, a transformation-competent cDNA library was created in the plant expression vector pER8-GW, carrying an estradiol-responsive expression cassette. Large-scale Arabidopsis transformation generated a collection of transgenic plants, each carrying a cDNA clone. Transgenic plants were screened for salt tolerance, ABA insensitivity or activation of reporter gene constructs. Several salt tolerant or ABA insensitive lines were obtained and characterized. In some lines reporter genes were activated upon the induction of transgene expression, in the absence of stress. In the line C38-33 increased salt tolerance was obtained by the activation of a full length cDNA, coding for a previously unknown protein with S1 domain. In the line ADH-121, activation of an AP transcription factor lead to the increased expression of the ADH-LUC reporter construct

Plants in Microgravity: Molecular and Technological Perspectives

Plants are vital components of our ecosystem for a balanced life here on Earth, as a source of both food and oxygen for survival. Recent space exploration has extended the field of plant biology, allowing for future studies on life support farming on distant planets. This exploration will utilize life support technologies for long-term human space flights and settlements. Such longer space missions will depend on the supply of clean air, food, and proper waste management. The ubiquitous force of gravity is known to impact plant growth and development. Despite this, we still have limited knowledge about how plants can sense and adapt to microgravity in space. Thus, the ability of plants to survive in microgravity in space settings becomes an intriguing topic to be investigated in detail. The new knowledge could be applied to provide food for astronaut missions to space and could also teach us more about how plants can adapt to unique environments. Here, we briefly review and discuss the current knowledge about plant gravity-sensing mechanisms and the experimental possibilities to research microgravity-effects on plants either on the Earth or in orbit

CRK5 Protein Kinase Contributes to the Progression of Embryogenesis of Arabidopsis thaliana

The fine tuning of hormone (e.g., auxin and gibberellin) levels and hormone signaling is required for maintaining normal embryogenesis. Embryo polarity, for example, is ensured by the directional movement of auxin that is controlled by various types of auxin transporters. Here, we present pieces of evidence for the auxin-gibberellic acid (GA) hormonal crosstalk during embryo development and the regulatory role of the Arabidopsis thaliana Calcium-Dependent Protein Kinase-Related Kinase 5 (AtCRK5) in this regard. It is pointed out that the embryogenesis of the Atcrk5-1 mutant is delayed in comparison to the wild type. This delay is accompanied with a decrease in the levels of GA and auxin, as well as the abundance of the polar auxin transport (PAT) proteins PIN1, PIN4, and PIN7 in the mutant embryos. We have previously showed that AtCRK5 can regulate the PIN2 and PIN3 proteins either directly by phosphorylation or indirectly affecting the GA level during the root gravitropic and hypocotyl hook bending responses. In this manuscript, we provide evidence that the AtCRK5 protein kinase can in vitro phosphorylate the hydrophilic loops of additional PIN proteins that are important for embryogenesis. We propose that AtCRK5 can govern embryo development in Arabidopsis through the fine tuning of auxin-GA level and the accumulation of certain polar auxin transport proteins

The AtCRK5 Protein Kinase Is Required to Maintain the ROS NO Balance Affecting the PIN2-Mediated Root Gravitropic Response in Arabidopsis

The Arabidopsis AtCRK5 protein kinase is involved in the establishment of the proper auxin gradient in many developmental processes. Among others, the Atcrk5-1 mutant was reported to exhibit a delayed gravitropic response via compromised PIN2-mediated auxin transport at the root tip. Here, we report that this phenotype correlates with lower superoxide anion (O-2(center dot-)) and hydrogen peroxide (H2O2) levels but a higher nitric oxide (NO) content in the mutant root tips in comparison to the wild type (AtCol-0). The oxidative stress inducer paraquat (PQ) triggering formation of O-2(center dot-) (and consequently, H2O2) was able to rescue the gravitropic response of Atcrk5-1 roots. The direct application of H2O2 had the same effect. Under gravistimulation, correct auxin distribution was restored (at least partially) by PQ or H2O2 treatment in the mutant root tips. In agreement, the redistribution of the PIN2 auxin efflux carrier was similar in the gravistimulated PQ-treated mutant and untreated wild type roots. It was also found that PQ-treatment decreased the endogenous NO level at the root tip to normal levels. Furthermore, the mutant phenotype could be reverted by direct manipulation of the endogenous NO level using an NO scavenger (cPTIO). The potential involvement of AtCRK5 protein kinase in the control of auxin-ROS-NO-PIN2-auxin regulatory loop is discussed

The mitogen-activated protein kinase 4-phosphorylated heat shock factor A4A regulates responses to combined salt and heat stresses

Heat shock factors regulate responses to high temperatures, salinity, water deprivation or heavy metals. Their function in stress combinations is however not known. The Arabidopsis HEAT SHOCK FACTOR A4A (HSFA4A) was previously reported to regulate responses to salt and oxidative stresses. Here we show, that the HSFA4A gene is induced by salt, elevated temperature and combination of these conditions. Fast translocation of HSFA4A-YFP protein from cytosol to nuclei takes place in salt-treated cells. HSFA4A can be phosphorylated not only by MAP kinases MPK3/6 but also by MPK4 and Ser309 is the dominant MAPK phosphorylation site. In vivo data suggest that HSFA4A can be substrate of other kinases as well. Changing Ser309 to Asp or Ala has altered intramolecular multimerization. Chromatin immunoprecipitation assays confirmed binding of HSFA4A to promoters of target genes encoding the small heat shock protein HSP17.6A and transcription factors WRKY30 and ZAT12. HSFA4A overexpression enhanced tolerance to individually and simultaneously applied heat and salt stresses through reduction of oxidative damage. Our results suggest that this heat shock factor is a component of a complex stress regulatory pathway, connecting upstream signals mediated by MAP kinases MPK3/6 and MPK4 with transcription regulation of a set of stress-induced target genes

AtCRK5 Protein Kinase Exhibits a Regulatory Role in Hypocotyl Hook Development during Skotomorphogenesis

Seedling establishment following germination requires the fine tuning of plant hormone levels including that of auxin. Directional movement of auxin has a central role in the associated processes, among others, in hypocotyl hook development. Regulated auxin transport is ensured by several transporters (PINs, AUX1, ABCB) and their tight cooperation. Here we describe the regulatory role of the Arabidopsis thaliana CRK5 protein kinase during hypocotyl hook formation/opening influencing auxin transport and the auxin-ethylene-GA hormonal crosstalk. It was found that the Atcrk5-1 mutant exhibits an impaired hypocotyl hook establishment phenotype resulting only in limited bending in the dark. The Atcrk5-1 mutant proved to be deficient in the maintenance of local auxin accumulation at the concave side of the hypocotyl hook as demonstrated by decreased fluorescence of the auxin sensor DR5::GFP. Abundance of the polar auxin transport (PAT) proteins PIN3, PIN7, and AUX1 were also decreased in the Atcrk5-1 hypocotyl hook. The AtCRK5 protein kinase was reported to regulate PIN2 protein activity by phosphorylation during the root gravitropic response. Here it is shown that AtCRK5 can also phosphorylate in vitro the hydrophilic loops of PIN3. We propose that AtCRK5 may regulate hypocotyl hook formation in Arabidopsis thaliana through the phosphorylation of polar auxin transport (PAT) proteins, the fine tuning of auxin transport, and consequently the coordination of auxin-ethylene-GA levels

The role of Arabidopsis genes involved in abiotic (osmotic, oxidative and gravitropic) stress response regulation

The warming of overall climate requires to breed plant cultivars tolerant to extreme osmotic tolerance, e.g. to high salt concentration in order to improve their chance to survive deleterious effects of abiotic stress conditions. Our initial aim is to isolate and characterize abiotic stress response regulatory genes arisen from Arabidopsis thaliana which is known as a model species for such investigations in higher plant. For this reason, a Ser/Thr protein kinase, the CRK5 was chosen. The CRK5 protein kinase is partly functionally characterized exhibiting a role in the regulation of gravitropic responses of Arabidopsis thaliana roots (Rigó et al., 2013). The CRK5 is a plasma membrane associated kinase that forms U-shaped patterns facing outer lateral walls of root epidermis cells. The CRK5 phosphorylates the hydrophilic loop of the auxin efflux transporter PIN2 in vitro. Thus, delayed gravitropic response of crk5 mutant reflects defective phosphorylation of PIN2 and deceleration of its brefeldin sensitive membrane recycling. Recently, we have been investigating the regulatory role of CRK5 protein kinase under osmotic (salt) and oxidative (hydrogen peroxide) stresses. The aim was to gather additional information regarding its role in the regulation of responses to either high salinity or oxidative stress, and consequently, regarding the impact on the auxin biosynthesis, transport and signaling. CRK protein kinase is hypothesized to be involved in the regulation of the effect of reactive oxygen species (ROS, e.g. hydrogen peroxide)